Test drugs
Gamithromycin (Luoyang Huizhong Animal Medicine Co., Ltd. Luoyang, Henan, China) was administered via injection at a dose of 150 mg/ml (15.0% w/v). tulathromycin (Draxxin, Zoetis) was administered as a control drug at a concentration of 100 mg/ml.
Animals
The study was conducted at Luzhou Gulin Langduoduo cattle farm, using 12-month-old shelf cattle sourced from various locations. At the farm, dedicated personnel diligently attend to the daily feeding, health, and hygiene requirements of the cattle.
Case selection and group
Inclusion and exclusion criteria
Before the study, a comprehensive clinical examination was performed on cattle with naturally occurring BRD, and their mental state and respiratory symptoms were scored according to the criteria listed in Table 1.
Table 1
Bovine Respiratory Disease BRD Symptom Scoring Criteria
Content | Judgment criteria | Score |
Depression | Normal | 0 |
Mild depression | 1 |
Moderate depression | 2 |
Severe depression | 3 |
Moribund | 4 |
Respiratory character | Normal | 0 |
Mild respiratory distress | 1 |
Moderate respiratory distress | 2 |
Severe respiratory distress | 3 |
To be enrolled, animals had to show signs of naturally occurring BRD as defined by the following criteria: being at least 15 days old, having a depression score or respiratory symptom score ≥ 1, and having a rectal temperature ≥ 40℃. Nasal swab samples were collected from sick cattle, and tests for detecting the pathogenic bacteria that cause BRD (P. multocida, M. haemolytica, etc.) were performed.
Animals that were debilitated, suffering from a systemic disease other than BRD, injured, moribund, milking or about to give birth and those with a known history of previous bacterial vaccinations for BRD or antimicrobials within 30 days of enrolment were excluded from the study.
Allocation and treatment
Cattle that met the enrolment criteria were grouped into two pens based on the order of the ear number, the subject group (group 1) and the control group (group 2), with 30 cattle in each group, and the severity of disease in each group was the same.
The cattle were weighed after clinical examination and qualification to ensure an accurate treatment dosage. Thirty cattle in each treatment group were single subcutaneously (SC) injected with either 6 mg/kg body weight (BW) gamithromycin (group 1) or 2.5 mg/kg BW tulathromycin (group 2). No other antibacterial or anti-inflammatory drugs were given to any test cattle throughout the trial period (from day 0 to day 14).
Clinical examination
From day 1 to day 14 of this study, a clinical assessment of the general appearance of the test animals (posture, behaviour, rectal temperature) and respiratory symptoms (type of breathing, respiratory frequency, coughing) was made. The results were transformed into a mental status score and a respiratory symptom score (Table 1). The injection site reactions were evaluated daily from day 1 to day 14.
Microbiological examination
On day 0, a nasopharyngeal swab sample was obtained from cattle (at least 10% of the test cattle) before dosing. If sick cattle died during the study, the carcass was necropsied, and lung tissue was taken for the isolation, culture, and identification of pathogenic bacteria. Nasal swab samples were taken on the day of withdrawal for the isolation and characterization of pathogens if cattle were withdrawn from the study due to failure to respond to BRD treatment.
Nasal swab samples were collected as follows: One nostril of the sick cattle was wiped clean with a disinfected paper towel. Then, a nasal swab sample was collected with a sterilized, long-handled cotton swab. Sampling records were made on the sick cattle record sheet, the sick cattle culling record sheet (invalidated for BRD treatment), the necropsy report, and the nasal swab record sheet collected on day 14 of the trial; nasal swabs were placed in test tubes containing 5% equine serum brain heart infusion (BHI) liquid medium (5 mL) and returned to the laboratory at low temperatures to be isolated for cultivation.
BRD-associated pathogens include P. multocida, M. haemolytica, H. somni, M. bovis, S. pneumoniae, Arcanobacterium pyogenes, Escherichia coli, Klebsiella pneumoniae, Enterococcus, Staphylococcus aureus, Pseudomonas aeruginosa, and Bacillus licheniformis, among others. These pathogens are also probable causes of the onset of BRD. Therefore, this experiment aimed to isolate and culture P. multocida, M. haemolytica, H. somni, Streptococcus, Trueperella pyogenes, E. coli, K. pneumoniae, Enterococcus, S. aureus, P. aeruginosa, and B. licheniformis using BHI agar medium (Beijing Luqiao) containing 10% sheep blood. Additionally, M. bovis was isolated and cultured using a pleuropneumonia-like organism (PPLO) solid medium (BD Company).
Bacteria were isolated, cultured, and subsequently identified utilizing the following methodology:
1) Isolation, culture, and identification of general bacteria: The test tube containing BHI liquid culture medium and the nasal swab was delivered to the laboratory, where it was shaken and mixed with a vortex device. The resulting suspension was picked up with an inoculation ring and marked on BHI agar medium containing 10% sheep blood. The culture dish was placed in an incubator containing 5%~10% CO2 and incubated at 37°C for 24 ~ 48 h. Typical colonies of different forms on the plate were selected for Gram-staining microscopy. According to the colony characteristics and Gram staining characteristics, a representative single settlement was established for pure culture, and the refined culture strains were amplified and sequenced by 16S rDNA. PCR-amplified 16S rDNA sequencing was performed under the following conditions: predenaturation at 94°C for 4 min; denaturation at 94°C for 30 s, annealing at 56°C for 30 s, and extension at 72°C for 90 s for 31 cycles; final elongation at 72°C for 10 min; and storage at 4°C. The upstream primer was 5ʹ-AGAGTTTGATCCTGGCTCAG-3ʹ. The downstream primer was 5ʹ -ACGGTTACCTTGTTACGACTT-3ʹ, and the target band of approximately 1500 bp was obtained for sequencing analysis. The bacterial species were determined comprehensively according to the characteristics of bacterial culture, morphology as determined by staining and visualized with microscopy, biochemical test results, and sequencing results.
2) Mycoplasma isolation, culture, and identification: Part of the nasal swab suspension was filtered with a 0.22 µm sterile filter. The appropriate amount was absorbed and coated on PPLO solid medium and incubated at 37°C in an incubator containing 5%~10% CO2 for 5 ~ 7 days. The growth status of the colonies on the tangible medium was determined under a microscope, and the suspected mycoplasma colonies were selected and transferred to liquid PPLO medium for culture. The culture was identified by PCR (PCR conditions: predenaturation at 94°C for 4 min; denaturation at 94°C for 30 s, annealing at 55°C for 30 s, and extension at 72°C for 30s for 35 cycles; final elongation at 72°C for 5 min; and storage at 4°C). The upstream primer was 5ʹ-GCGCAGTGCTGATGTTGAAT-3ʹ. The downstream primers were 5ʹ-ACAAAAATCAATAGGAAAGCACCCT-3ʹ, and the target band size was 529 bp.
Criteria for evaluating therapeutic effects
Within 14 days of drug administration, the efficacy of the test drug and the control drug were judged according to the following criteria: body temperature, spirit, respiratory symptoms, and other clinical manifestations.
Ineffectiveness
1) Treatment was considered ineffective if the sick cattle met the following criteria on two consecutive days from day 2 to day 14 of dosing: mental depression score ≥ 1, respiratory symptom bottle score ≥ 1, and rectal temperature ≥ 40 ºC.
2) From day 2 to day 14 of dosing, treatment was judged to be ineffective if sick cattle had to be withdrawn from the study due to severe BRD and given other medications or they died due to BRD.
Ineffective rate (%) = (number of sick cattle in the group for which treatment was ineffective /number of sick cattle in the group for which the drug was used) × 100
Effective
From the 2nd day to the 14th day of administration, the sick cattle were not judged as treatment was not ineffective, but spirit, respiration, and rectal temperature still had not fully returned to normal, and treatment was considered effective.
Effective rate = [(number of sick cattle in the group for which treatment was effective + number of sick cattle in the group that were cured)/number of medicated sick cattle in the group] × 100
Cured
From the 2nd day to the 14th day of administration, sick cattle for which spirit, respiration, and rectal temperature returned to normal and the disease did not recur for 7 consecutive days were judged to be cured.
Cure rate = (number of cured cattle in the group/number of medicated cattle in the group)
Statistical analyses
Statistical analysis was performed using SPSS 13.0 Comparisons between experimental groups were performed using a two-sided test with 5% significance levels. Mental state scores, rectal temperatures, and respiratory symptom scores were expressed as the mean ± standard deviation (± SD), and the Mann‒Whitney test was used to compare the differences between groups, while the Wilcoxon signed rank test was used to compare differences within groups. The efficiency rate and cure rate were expressed as percentages (%), and the differences between groups were tested by Fisher’s exact test.